Characterisation of Caco3 Phases During Strain-Specific Ureolytic

www.nature.com/scientificreports OPEN Characterisation of CaCO3 phases during strain-specifc ureolytic precipitation Alexandra Clarà Saracho1 ✉ , Stuart K. Haigh1, Toshiro Hata2, Kenichi Soga3, Stefan Farsang4, Simon A. T. Redfern5 & Ewa Marek1 Numerous microbial species can selectively precipitate mineral carbonates with enhanced mechanical properties, however, understanding exactly how they achieve this control represents a major challenge in the feld of biomineralisation. We have studied microbial induced calcium carbonate (CaCO3) precipitation (MICP) in three ureolytic bacterial strains from the Sporosarcina family, including S. newyorkensis, a newly isolated microbe from the deep sea. We fnd that the interplay between structural water and strain-specifc amino acid groups is fundamental to the stabilisation of vaterite and that, under the same conditions, diferent isolates yield distinctly diferent polymorphs. The latter is found to be associated with diferent urease activities and, consequently, precipitation kinetics, which change depending on pressure-temperature conditions. Further, CaCO3 polymorph selection also depends on the coupled efect of chemical treatment and initial bacterial concentrations. Our fndings provide new insights into strain-specifc CaCO3 polymorphic selection and stabilisation, and open up promising avenues for designing bio-reinforced geo-materials that capitalise on the diferent particle bond mechanical properties ofered by diferent polymorphs. Calcium carbonate (CaCO3) makes up almost 4% of the Earth’s crust and has been studied extensively due to its importance in biomineralisation in natural environments, including carbon cycling, alkalinity generation, and the biogeochemical cycling of elements1–3. In addition to its importance in nature, however, the enhanced mechanical properties of certain biotic calcium carbonates have inspired many studies to try to understand their structural secrets4–6, and prompted their use in geotechnical engineering to improve the mechanical response of soils7–11. A clear example of these natural materials is nacre in mollusc shells, formed of microlaminate compos- ites of aragonite and/or calcite, each with an associated organic matrix that gives them a fracture toughness 3000 times greater than that of the constituent mineral alone12. Clearly, the coupling of a mineral phase with an organic material (i.e. biomineral) plays a vital role in the formation and stabilisation of the fnal CaCO3 precipitate, in addition to contributing to its ultimate mechanical properties4,13–17. However, understanding the interrelationship between biotic precipitation, polymorphism, and long-term stabilisation has proven to be far from trivial. It is well established that CaCO3 has three polymorphs: vaterite, aragonite, and calcite, with rhombohedral, orthorhombic and hexagonal structures respectively, in order of decreasing solubility and increasing thermodynamic stability18. Additional metastable forms have been noted in the literature, all of which are hydrated: monohydrocalcite (CaCO3·H2O), ikaite (CaCO3·6H2O), calcium carbonate hemihydrate (CaCO3·1/2H2O), and amorphous calcium carbonate (ACC)19,20. Tus far, most studies have tackled the formation and crystallisation of CaCO3 in abiotic systems, with a particular focus on ACC and vaterite as intermediates in the crystallisation of 21–25 CaCO3 . However, in contrast to the unique nature of the equilibrium state, multiple reaction pathways from a given initial condition to that fnal state of thermodynamic equilibrium may exist. Such reaction pathways can be very sensitive to minor impurities and environmental perturbations, such as the presence of microorganisms, which modify the energy barrier from reactant to product phases26. It has even been suggested that organic macromolecules associated with bacterial activity cause the Ostwald step sequence to stop at one of its inter- mediate stages: ACC → vaterite → calcite21, making biotic vaterite precipitation far more common than would 1Department of Engineering, University of Cambridge, Cambridge, CB2 1PZ, UK. 2Department of Engineering, Hiroshima University, Hiroshima, 739-8527, Japan. 3Department of Engineering, University of California-Berkeley, California, 94720, Berkeley, USA. 4Department of Earth Sciences, University of Cambridge, CB2 3EQ, Cambridge, UK. 5Asian School of the Environment, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore. ✉e-mail: [email protected] SCIENTIFIC REPORTS | (2020) 10:10168 | https://doi.org/10.1038/s41598-020-66831-y 1 www.nature.com/scientificreports/ www.nature.com/scientificreports be anticipated in abiotic systems. From a chemical standpoint, the polymorphous composition is governed by the addition of the reactants, which lead to a supersaturation state in which the concentration of calcium and 18,27 carbonate ions exceed the solubility product of CaCO3 . Tis simultaneously triggers the nucleation of crystals and the dissolution of the colloidal ACC precursor18. However, local variations of the calcium and carbonate ion activity product (IAP) and thus saturation, can favour the formation of one polymorph over another. Vaterite, for example, typically precipitates in highly supersaturated and moderately alkaline environments14,21,23. Within this context, enzymatic hydrolysis of urea presents a straightforward process to understand the precise role of microbes in microbial induced calcium carbonate precipitation (MICP). Tis is because the urease enzyme is ubiquitous in microorganisms, yeast, and plants5,28. In addition, it can be easily induced using inexpensive chemicals and is the most widely used process in biomediated soil improvement applications. Ureolytic bacteria + enzymatically hydrolyse urea (CO(NH2)2), resulting in the production of ammonium (NH4 ) and dissolved inor- ganic carbon (DIC), which in turn increase pH and favour CaCO3 precipitation in the presence of soluble calcium ions (eq. 1). Tis study focuses on three diferent ureolytic bacterial strains, all belonging to the Sporosarcina species: Sporosarcina pasteurii (ATCC 11859), Sporosarcina aquimarina (ATCC BAA-723), and Sporosarcina newyorkensis–a newly isolated microbe from the deep sea in ofshore Japan extracted by the National Institute of Advanced Industrial Science and Technology (AIST) using pressure-core nondestructive analysis tools29. To our knowledge, this has never been studied before. Tese strains were selected as they proliferate in diferent isolation environments: surface dry conditions, and shallow and deep sea, respectively. +−2 CO(NH)22+→2H24O2NH + CO3 (1a) 22−+ CO3 +→Ca CaCO3↓ (1b) Our aim is to shed light on the interaction between the components of mineralised biological materials involved in CaCO3 precipitation–namely minerals, macromolecules and water–, and understand their infuence on the stabilisation of diferent CaCO3 polymorphs. In addition, we wish to understand how strain-specifc precipitation kinetics promote and afect this process. For this purpose, CaCO3 was precipitated in vitro in 14 mL test tubes via the three diferent ureolytic soil bacteria described above. To ensure analogous reference conditions, strains were cultivated under sterile conditions at the same pressure and temperature–i.e. P = Patm and T = 30 °C–to an optical density (OD600) of ~0.5, and subsequently subject to identical external treatment conditions. Te cementation treatment liquid medium consisted of a premixed solution of an equimolar amount −1 of urea and CaCl2 (0.3 M), in addition to 3 gL of Nutrient Broth, all dissolved in deionised water. All samples were created using a ratio of bacteria solution to cementation solution of 1:2. Results strongly suggest that the presence of structural water together with specifc amino acids is fundamental to the stabilisation of vaterite and that, at the same initial OD600 and treatment conditions, diferent strains yield distinctly diferent polymorphs. For this reason, we compared the precipitation kinetics, and the pressure-temperature dependence of bacterial population and urease activity for the three microorganisms. Finally, S. pasteurii–which is the most common soil bacterium used in geotechnical engineering applications–was also investigated under varying urea-CaCl2 solution concentrations and initial OD600. Te mineralogy, morphology, and properties of precipitates were char- acterised using an array of complementary techniques, namely thermogravimetric analysis coupled with mass spectroscopy (TGA-MS), Raman spectroscopy (RM), X-ray powder difraction (XRD), and scanning electron microscopy (SEM); and the precipitation kinetics of the three microorganisms quantifed through measurement 2+ of calcium ion (Ca ) concentrations and pH (Table S4). Ultimately, our results suggest that strain-specifc CaCO3 precipitation occurs during ureolytic MICP, possibly due to diferences in the urease enzyme, and its response to treatment concentrations and pressure-temperature variations, and that CaCO3 polymorphism in biotic systems is far more common than previously anticipated. Tis may have signifcant implications for biomediated soil improvement systems. Results Amorphous and crystalline CaCO3 polymorphs. XRD analysis (Fig. 1a) revealed that calcite was the primary polymorph that precipitated in the presence of S. newyorkensis (SN01-0.3M), along with small traces of halite resulting from the drying of the marine broth media used to cultivate the isolate30 (Fig. S1). On the other hand, precipitates of S. aquimarina (SA01-0.3M)

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